Sciex analyst work station software version 1.6.3

For widely targeted metabolomics, Sciex analyst work station software Version 1.6.3 (Sciex, Framingham, MA, USA) was employed for MRM data acquisition and processing. MS raw data files were converted to the TXT format using MSconventer. R software Version 4.0.0 (https://www.r-project.org/ (accessed on 19 September 2020)) was used for peak detection and annotation. For untargeted metabolomics, the acquired mass spectral raw data were imported into Compound Discoverer 3.1 (Thermo Scientific, Wilmington, NC, USA) for data processing, mainly including peak extraction, retention time correction, adduct ion merging, missing value filling, and background peak labeling. Finally, the compound molecular weight, retention time, peak area, and identification results were exported. PCA, PLS-DA, and 200 permutation tests were performed using SIMCA software Version 14.1 (Umetrics AB, Umeå, Sweden). The metabolic pathway analysis of the differentially expressed metabolites was performed by the online tool MetaboAnalyst (https://www.metaboanalyst.ca/ (accessed on 11 March 2022)).

After the AT sample was pretreated [64 (link)], an EXIONLC System was used for UHPLC separation (Sciex). Formic acid at 1% in water served as mobile phase A, and acetonitrile served as mobile phase B. The temperature in the column was fixed at 40 °C. The injection volume was 2 L, and the autosampler’s temperature was set at 4 °C. For the development of the test, a Sciex QTrap 6500+ (Sciex Technologies) was used. The typical ion source settings were as follows: ion spray voltage: +5500/−4500 V; curtain gas: 35 psi; temperature: 400 °C; ion source gas 1:60 psi; ion source gas 2:60 psi; and DP: 100 V. Finally, SCIEX Analyst Work Station Software (Version 1.6.3) was employed for MRM data acquisition and processing. An in-house R program and database were applied for peak detection and annotation.

The UHPLC–MS analysis was carried out using EXIONLC System (Sciex, Framingham, MA, USA) by Shanghai Hoogen Biotech, Shanghai, China using the parameters as described previously [31 (link)]. The mobile phase A contained 0.1% formic acid in H₂O (v/v), and mobile phase B was acetonitrile (Merck KGaA, Darmstadt, Germany); column temperature: 40 °C; auto-sampler temperature: 4 °C; injection volume: 2 μL. Typical ion source parameters were: IonSpray voltage: +5500/−4500 V; curtain gas: 35 psi; temperature: 400 °C; ion source Gas 1:60 psi; ion source Gas 2: 60 psi; and declustering potential (DP): ±100 V. The SCIEX Analyst Work Station Software (Version 1.6.3) was employed for multiple reaction monitoring (MRM) data acquisition and processing. In-house R program and database were applied for peak detection and annotation (Shanghai Hoogen Biotech, Shanghai, China).